AU8103687A

AU8103687A - Improved nucleic acid hybridization technique and kit therefor

Info

Publication number: AU8103687A
Application number: AU81036/87A
Authority: AU
Inventors: Joan Marlyn Beebe; Linda Lee Glanville; Jeffry Joseph Leary; Edward Gray Rice
Original assignee: Beckman Instruments Inc
Current assignee: Beckman Coulter Inc
Priority date: 1986-10-14
Filing date: 1987-10-06
Publication date: 1988-05-06
Also published as: NO882593D0; WO1988002785A3; JPH01501339A; FI882800A0; DK324088D0; CA1309932C; FI882800A; WO1988002785A2; NO882593L

Description

IMPROVED NUCLEIC ACID HYBRIDIZATION TECHNIQUE

AND KIT THEREFOR

BACKGROUND

The present invention relates to a nucleic acid hybridization procedure, and a diagnostic kit for use with the procedure.

All of the articles and references identified in this specification are incorporated herein by reference.

In traditional microbial diagnostics the presence of a microbe in a sample is determined by isolating the microbe. After enrichment cultivations, the microbe is determined either on the basis of its biochemical properties or its immunological properties. Both methods of identification require that the microbe in the sample be able to propagate. Such identification can be laborious and very time consuming. With the development of genetic research, it was found that microbes, even ones that are no longer viable, can be determined by the specific nucleic acids they contain. The nucleic acid may be deoxyribonucleic acid (DNA), which is usually double stranded, or ribonucleic acid (RNA), which is usually single stranded. Organisms such as bacteria, fungi, viruses, yeasts, etc., can all be thus determined. Generally the identification procedure includes artificially induced lysis, whereby the cell wall of the organism is ruptured to release the nucleic acid, which can then be determined .

The hydrogen-bonded structure of the double helix of a nucleic acid molecule (e.g. DNA) can be disrupted by heating and/or by treatment with alkali. Because there are no covalent bonds connecting the partner strands, the two polynucleotide chains of a duplex, micleic acid molecule separate entirely when all the hydrogen bonds are broken. This process of strand separation is called denatur-ation. An extremely useful property of denatured nucleic acids is that, under appropriate conditions, the reaction can be reversed, so that two separated complementary strands from the same source can reform into a double helix. This is called renaturation. Renaturation involves the reaction of two complementary nucleotide sequences that were separated by denaturation. This technique can also be extended to allow any complementary nucleotide sequences to anneal with each other to form a duplex structure. This is generally referred to as hybridization when nucleotide sequences from different nucleic acid moieties are involved; e.g., reaction between single-stranded DNA and RNA, or between two single- stranded DNA's from different sources.

Nucleic acid hybridization is a well known method for identifying specific nucleic acids. This ability of two single-stranded nucleic acid preparations to hybridize forms the basis of current nucleic acid assays. The principle of such assays is to expose two single-stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that is formed.

Hybridization assays usually involve the use of polynucleotide hybridization probes. A probe generally comprises a single-stranded fragment of a nucleic acid, or a double-stranded fragment denatured. It is characterized by having a specific nucleotide sequence which is complementary to a corresponding nucleotide sequence on the target nucleic acid (the nucleic acid to be determined). The probe and the target polynucleotide (rendered single-stranded), when brought together, form duplex molecules by base pairing of the complementary sequences. The probe is generally prepared in purified reagent form, and a readily detectable label can be incorporated into the molecular structure of the probe. The presence of the target polynucleotide can thus be confirmed by the formation of duplex hybrid molecules carrying the label.

Polynucleotide hybridization probes offer inexpensive, efficient, and rapid means for detecting, localizing, and isolating "target" nucleotide sequences. Klausner et al., Biotechnology, August 1983:471-478, provide interesting background on polynucleotide hybridization probes, including a discussion of their preparation and use. Known methods for preparing polynucleotide hybridization probes and for using such probes are well documented in the literature. . See, for example. Southern, J. Mol. Biol. 98:503-517 (1975); Falkow et al., U.S. Patent No. 4,385,535; Leary et al., Proc. Natl. Acad. Sci. 80:4045-4049 (1983); Langer-Safer et al., Proc. Natl. Acad. Sci. 79.4381-4385 (1982); and Langer et al., Proc. Natl. Acad. Sci. 78:6633- 6637 (1981). As disclosed by these and other references, such known methods for preparing probes typically comprise cloning a probe region into a double stranded DNA plasmid. The plasmid carrying the probe region is labeled, typically by enzymatic polymerization techniques. Such techniques include, for example, nick translation (Rigby et al., J. Mol. Biol. 113:237 (1977)); gap-filling (Bourguignon et al., J. Virol. 20:290 (1976)); and terminal addition, which techniques are carried out in the presence of modified nucleotide triphosphates. The probe nucleic acid can also be labeled by chemical means with haptens or biotin (Tchen et al, P.N.A.S. 81:3466 (1984), Forster et al., Nucl. Acids Res. 13:745 (1985), Viscidi et al., J. Clin. Microbiol. 23:311 (1986).

There are two principal ways of performing hybridization assays: solution (or liquid) hybridization, and solid carrier hybridization. In solution hybridization, single-stranded nucleic acid preparations are mixed together in solution. For larger amounts of material, the reaction can be followed by the change in optical density. When smaller amounts of material are involved, the probe may carry a readily detectable label, such as a radioactive label. The unreacted single strands are separated from the double-strands, and the double-stranded nucleic acid can be determined by detecting the presence of the label in the double stranded material.

Hydroxyapatite (HA) has been used as a standard method for separating hybridized probe from non-hybridized probe in solution. Under the proper conditions, HA can selectively bind hybridized DNA probe, but does not bind non-hybridized probe. Other methods are also available for separating hybridized probe from non-hybridized probe. One such method involves the use of a specific enzyme, e.g. S₁, nuclease, to selectively degrade non-hybridized probe to smaller fragments. The double-stranded molecules are not affected by the enzyme. The degraded probe can then be separated by well known size separation techniques, such as precipitation with polyethylene glycol, chromatography, electrophoresis, and ultracentrifugation, etc. Britten et al. Methods in Enzymology, XXIX, page 363, Eds., Grossman & Moldave, Academic Press, New York, 1974.

Solution hybridization has the advantages of speed and reaction efficiency. However, it also has serious drawbacks. The steps for separating hybridized from non- hybridized probe are generally labor intensive, time consuming, do not lend themselves to automation, and may otherwise be limiting. For example, the size separation techniques described above are relatively non-specific, inefficient, and show poor reproducibility. Although the HA separation method is more specific, it is generally useful only if the target polynucleotide is present in large excess over the probe, or if the target is RNA, or both. Further, with unpurified samples, e.g. plant and animal tissue homogenates, blood, feces, nasal and urethral mucous, etc., effective separation can be very difficult.

In solid-carrier hybridization, one of the singlestranded nucleic acid preparations is immobilized by being affixed on a solid-carrier. Numerous methods exist for coupling either terminal end of a polynucleotide to a given support. See, e.g. Weissback, A. and Poonian, M., Methods in Enzvmology, Vol. XXXIV, Part B, 463-475, 1974. Also, derivative forms of polynucleotides, for example, one carrying a terminal aminohexyl nucleotide, can be easily attached on a variety of supports. Mosbach, K., et al., Methods in Enzymology, Vol. XLIV, 859-886, 1976. Oligoribonucleotides may be immobilized on btsronate derivatives of various supports. Schott, H., et al., Biochemistry, 12, 932, 1973.

As an example, nitrocellulose adsorbs singlestranded DNA, but not RNA. Moreover, further adsorption o f DNA on the ni trocellulose can be prevented by well known treatments. Then if a second denatured DNA, or RNA, preparation is added, it will become affixed to the solid- carrier only if it is able to base pair with the DNA that was originally adsorbed. Usually, the nucleic acid preparation which is not originally bound to the solid- carrier is labeled. After the hybridization reaction is completed, the solid carrier is separated from the reaction mixture, and the degree of hybridization can be determined by measuring the label affixed to the solid-carrier.

A refinement of solid-carrier hybridization is sandwich hybridization, such as that discussed in Ranki, U.S. Patent No. 4,486,539. The sample is subjected to conditions which render the target polynucleotide (the nucleic acid to be determined) single-stranded. The sample is then mixed with two purified nucleic acid reagents. The first nucleic acid reagent comprises a single stranded fragment of nucleic acid, having a nucleotide sequence of at least 10 bases, and being affixed to a solid-carrier. The second nucleic acid reagent comprises a single stranded fragment of nucleic acid, having a nucleotide sequence of at least 10 bases, and being labeled with a radioisotope. The nucleic acid reagents are capable of forming hybrid molecules by complementary base pairing with the target polynucleotide. The two nucleic acid reagents are not capable of hybridizing with each other. The solid carrier is then washed to substantially remove the label which is not incorporated in the hybrid molecules. The presence of the target nucleic acid is then determined by measuring the label on the washed solid carrier. Sandwich hybridization also provides improved specificity over methods using a single nucleic acid reagent, because it involves two specific hybridization processes.

An advantage of solid-carrier hybridization is the ease with which the hybrid molecule formed from the target nucleic acid and the probe(s) can be separated from the reaction mixture. However, prior art solid-carrier hybridization techniques, including the sandwich hybridization procedure of Ranki discussed above, suffer a serious drawback. Kinetics dictate much slower reaction rates in solid-carrier hybridization than in solution hybridization, as not all the hybridization components are allowed to diffuse. A reaction that takes minutes in solution can take hours, even days, to complete when a solid-carrier is involved. Moreover, the hybridization efficiency is also much lower, since some nucleic acids are unavailable for base pairing. Further, the degree of preparation required is substantial! Usually several hours are required to prepare the sample for hybridization, and one to two hours are required for washing. Relatively large amounts of probes are also required.

It is desirable to have a method for assaying nucleic acid which combines the advantages of both solution hybridization and solid carrier hybridization.

Genetic disease diagnosis, in the present state of the art, generally involves time consuming and labor intensive techniques. A currently accepted diagnostic method for the determination of genetic disease is a procedure known as restriction fragment length polymorphization (RFLP). RFLP technology generally employs a somewhat tedious separation step based on the size of the genetic pieces produced pursuant to treatment of sample DNA with a restriction enzyme. The specialized case of sickle- cell anemia presents the most easily understood variation of RFLP technology. For example. The New England Journal of Medicine, July, 1982: pp. 30-32, and pp. 32-36 contains two articles which illustrate how the current prenatal test for sickle-cell anemia is performed. Generally the test sample DNA is treated with a restriction enzyme (Mst II) which produces two different sized pieces of the globin gene for the normal and sickle alleles. The pieces are separated by size, for example on an agarose electrophoresis gel. The pieces are then rendered single-stranded, transferred to a sheet of filter paper, and the exact pieces determined in the morass of similarly-sized pieces by a hybridization probe. Both the gel electrophoresis and transfer steps of the test require the use of highly skilled personnel, and are time consuming.

What is needed is a simplified method for diagnosing the presence of gene mutation due to genetic diseases.

SUMMARY

The present invention satisfies the above needs. Specifically, it covers a method for detecting, either qualitatively or quantitatively, a single-stranded target polynucleotide in a liquid sample. The method comprises the steps of:

(a) combining the sample with at least two different probes, being a first probe and a second probe, to form a reaction mixture, each probe comprising a single- stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the probes together forming hybrid molecules by complementary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the probes do not hybridize with each other, and the second probe having a detectable label;

(b) subsequently contacting the reaction mixture with a solid carrier which binds the first probe but not the second probe, and not the target polynucleotide; and

(c) subsequently determining if any of the label is bound on the solid carrier.

The single-stranded target polynucleotide can be formed by denaturing a double-stranded polynucleotide. Preferably the two probes bind different portions on the target polynucleotide to be determined, so that the two probes do not compete. Preferably the two different portions are adjacent or close by.

The method of this invention is suitable for microbial diagnostics. It is also suitable for genetic disease diagnosis such as that for sickle cell anemia, and for cancer diagnosis, among other uses. In this latter aspect, the method of the present invention incorporates a severing step, such as by the use of a restriction enzyme, together with a sandwich hybridization procedure. For example, the method can be used to detect a target polynucleotide in a sample which may also contain a standard polynucleotide, the nucleotide sequences of the two polynucleotide being substantially similar, the standard polynucleotide having a portion A and a portion B, the target polynucleotide having a portion A' and a portion B', the standard and target polynucleotides differing in at least one nucleotide which is located between portions A and B on the standard polynucleotide and between positions A' and B' on the target polynucleotide, the method comprising the steps of:

(a) severing both the standard and target polynucleotides present into single-stranded segments, such that none of the segments of the standard polynucleotide contains both portions A and B, while at least one of the segments of the target polynucleotide contains both portions A' and B';

(b) combining the treated sample from step (a) with at least two probes, being a first probe and a second probe, to form a reaction mixture, each probe comprising a single-stranded polynucleotide, the probes being unable to hybridize with each other, the first probe complementary base pairing with portion A, and with portion A', the second probe complementary base pairing with portion B, and with portion B', the two probes together forming hybrid molecules by complementary base pairing with the single-stranded segment of the target polynucleotide which contains both portions A' and B'; and

(c) subsequently determining the presence of hybrid molecules containing both of the probes.

Preferably neither probe is affixed to a solid carrier, the second probe has a detectable label, the step for determining the persence of hybrid molecules containing both of the probes further comprising the steps of:

(a) contacting the reaction mixture with a solid carrier which binds the first probe but not the second probe, and not segments containing only portion B or only portion B '; and (b) subsequently determining if any of the label is bound on the solid carrier.

DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

Fig. 1 is a schematic representation of the steps of the assay method of the present invention.

Fig. 2 is a schematic representation of a sickle cell assay using the method of the present invention.

Figs. 3 and 4 are plots of the relative signal of label detection vs. the target DNA concentration.

Fig. 5 is a plot of the binding characteristics of an avidin-cellulose solid carrier and a biotin-labelled probe.

DESCRIPTION

A method and kit including features of the present invention can be used to detect the presence of polynucleotide (such as DNA or RNA) containing organisms, such as viruses, bacteria, fungi, yeasts, other microorganisms, and other infectious agents. The method and the kit can be used, for example, in food hygiene investigations, medical diagnostic applications, and any microbial diagnostics. Suitable samples include animal and plant tissue homogenates, blood, serum, feces, nasal and urethral mucous, water, dust, soil, etc. Solid samples are first slurried or homogenized in a liquid medium to form the test sample. The method is also suitable for diagnosing genetic disease where normal genes have been caused to mutate. The method is also suitable for cancer diagnosis.

In the method of the present invention, a sample containing cells suspected to contain the polynucleotide to be detected (target polynucleotide) is pretreated, as necessary, to release the target polynucleotide from the cells of the organism into solution, or to render the cell wall permeable to the reagents used for detecting the target polynucleotide. The target pdlynucleotide is ususally a DNA or a RNA, or derivatives or fragments thereof.

Hybridization takes place between single stranded polynucleotides. Thus if the target polunucleotide is double stranded, the test sample is then subjected to conditions capable of denaturing the target polynucleotide present, e.g., heat, or heat plus high pH, such as 100 °C for 5 min., or treatment with 0.5 molar NaOH for 5 min. at 20-40°C. Conditions for the denaturation of polynucleotides are well known.

The method of the present invention, as illusrated in Fig. 1, comprises a solution sandwich hybridization step and a harvesting step. In the solution sandwich hybridization step, the test sample is combined with at least two different specific nucleotide hybridization probes to form a reaction mixture. For example, two probes can be used, being a first probe - a binding probe, and a second probe - an identification probe. Each probe comprises a single-stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the single-stranded target polynucleotide. Thus each probe is capable of complementary base pairing with the corresponding nucleotide sequence on the target polynucleotide. Preferrably the two probes bind different portions on the target polynucleotide, so that the two probes do not compete; preferably the portions are closely spaced apart (no more than about 300 nucleotides apart), more preferably the portions are immediately adjacent to each other (no more than about 10 nucleotides apart). The probes are incapable of hybridizing with each other. In the reaction mixture, the two probes and the single-stranded target polynucleotide together hybridize to form complex double-stranded hybrid molecules. To ensure fast and efficient hybridization, neither probe is affixed to a solid carrier. The second probe, the identification probe, is further characterized by having a readily detectable label. The procedure is called sandwich hybridization because the target polynucleotide is "sandwiched" between the two probes in the resulting hybrid molecules.

The probes can be contained in separate reagents. Because the probes do not hybridize with each other, it is also possible to have all the probes in a single reagent. The order of combining the probes with the test sample is generally not important. The probes can be added to the test sample in seriatum (one after another), all at the same time, or in any other combination.

The reaction mixture is maintained at conditions conducive to hybridization. The conditions depend on, and are generally known for, the particular polynucleotide moieties involved. The hybridization is allowed to go to substantial completion. For most polynucleotides this takes no more than about one hour. For example, at a salt concentration of abαut 0.15 molar, a temperature of 65°C, and a plasmid probe concentration at 1.0 microgm/ml, the^' time to reach 1/2 completion is less than 20 minutes.

In the harvesting step, the reaction mixture is contacted with a solid carrier capable of binding the first probe (the binding probe), but not the second probe (the identification probe), and not the target polynucleotide. The terms "bind" and "binding" herein shall refer to strong binding via specific functional groups, in contrast to low level non-specific binding. The hybrid molecules formed from the binding of the target polynucleotide to the two probes are thus bound on to the solid carrier via the binding probe.

The second probe, is chosen such that there is little non-specific binding of the second probe itself on to the solid carrier. The presence of the target polynucleotide in the test sample is thus confirmed by determining if any of the label is bound on the solid carrier. Quantitative determination of the amount of the target polynucleotide in the test sample is also possible, by measuring the actual amount of label bound on the solid carrier.

There are at least two basic methods for determining if any of the label is bound on the solid carrier, both measuring the distribution of the second probe in the solid and lquid phases of the reaction mixture after the harvesting step. In the first method, the excess identification probe not incorporated in the hybrid molecules is removed by conventional methods such as washing, aspiration, decantation, etc. The amount of label bound on the solid carrier is then measured. The presence of the label on the solid carrier indicates the presence of the target polynucleotide in the sample. In the second method, the solid carrier can be separated from the liquid phase of the reaction mixture, or it can remain mixed with the liquid phase. The amount of unbound second probe in solution in the liquid phase is measured and is compared to the total amount of second probe added to the reaction mixture. A difference in the two amounts indicates that some of the second probe is bound on to the solid carrier, which in turn indicates the presence of the target polynucleotide in the sample.

Preferably the first probe (binding probe) comprises a first binding group and the solid carrier comprises a second binding group, the two binding groups binding each other by forming strong bonds with each other rapidly. The two binding groups together constitute a binding pair. One of the binding groups of the binding pair is linked to the base material of the solid carrier, and the other binding group is part of the first probe. The binding pair can, for example, be any one of the following pairs: avidin-biotin, hapten-antibodies, antibodies-antigens, carbohydrates-lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzyme- substrate, boronates-cis dioles, staph A proteins- antobodies, enzymes-inhibitors, etc. The binding pairs also include pairs of derivatives of the above moieties. The criteria for choosing the particular binding pairs include (1) the speed with which the binding groups of the pair react to bind each other, (2) the strength of the bond between the binding groups of the pair, (3) minimal interference of the binding group on the first probe with the solution sandwich hybridization step, (4) ease with which the binding groups can be linked to the base material of the solid carrier, and be used to form the first probe.

The base material of the solid carrier can be of a material selected from, for example, agarose, cellulose, glass, latex, polyacrylamide, polycarbonate, polyamide (e.g. Nylon _TM ), polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof. This list is by no means exhaustive. Any solid on which one of the binding groups of the binding pair can be affixed is suitable.

The solid carrier can be in various forms. For example, it can be in the form of micro-particulates with particle sizes less than 100 microns (for example, mirocrystaline cellulose), macro particulates with particle sizes of 0.1 to 2.0 mm, sheets, tubes, pipet tips, plates, filters and beads, etc.

Methods for affixing the second binding group to the solid carrier are well known. Methods for derivatizing the polynucleotide moiety of the first probe to contain the first binding group are also also known. (Tchen et al., P.N.A.S. 81:3466 (1084), Forster et al., Nucl. Acids Res. 13:745 (1985), Viscidi et al., J. Clin. Microbiol. 23:311 (1986). Biotechnology, August 1983:471-478, J. Mol. Biol. 98:503-517 (1975); Falkow et al., U.S. Patent No. 4,385,535; Leary et al., Proc. Natl. Acad. Sci. 80:4045-4049 (1983); Langer-Safer et al., Proc. Natl. Acad. Sci. 79:4381-4385(1982); and Langer et al., Proc. Natl. Acad. Sci. 78:6633- 6637 (1981), Rigby et al, J. Mol. Biol. 113:237 1977), Bourguignon et al., J. Virol. 20:290 (1976).

The second probe (the identification probe) contains a readily detectable label. Various methods for labeling specific polynucleotide probes are known. Any label capable of being readily detected and which does not unduly interfere with the solution hybridization step, can be used. Suitable labels include radioisotopes, light-labels, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, etc., and their derivatives. Detection can be direct, as with radioisotopes, or indirect, as with a hapten followed by an enzyme-labeled antibody.

Radioisotopes such as ^{3 2}p, ¹²⁵I, etc., can be used to label the probe. The radio-labels can be detected by well known methods such as gamma counting or scintillation counting. However, the use of radioisotope labels can be expensive and hazardous. Detection of radioactivity generally requires expensive equipment. Special training for personnel and safety precautions are required for handling radioactive material. Moreover, radioisotopes have finite half-lifes; and thus the labeled polynucleotide probe usually has a relatively short shelf life (usually in the order of weeks).

For the above reasons, other label systems were developed, such as light-labels and enzyme-action based labels. As discussed in Heller, et al., European Patent Application No. 82303701.5, light-labels can be chemiluminesceήt, bioluminescent, fluorescent, or phosphorescent, and under the proper conditions can provide sensitivities comparable to that of-radioisotopes. The light-label can be attached to any point on the single- stranded polynucleotide segments of the probe; however, terminal positions are known to be more desirable.

Some examples of light-labels are as follows: (1) chemiluminescent: peroxidase and functionalized iron porphyrin derivatives, (2) bioluminescent: bacterial luciferase, firefly luciferase, flavin mononucleotide (FMN), adenosine triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and various long chain aldehydes (decyl aldehyde, etc.); (3) fluorescent: fluorescent nucleotides such as adenosine nucleotides, etheno-cytidine nucleotides, etc., or functionalized nucleotides (amino-hexane adenosine nucleotides, murcurinucleotides, etc.), which can first be fl uorescently labelled, and then covalently attached to the single- stranded polynucleotide segment of the probe; (4) phosphorescent: 2-diketones such as 2,3 butadione, 1- [carboxyphenyl]-1,2-pentanedione, and 1-phenyl-1,2- propanedione. Means for attaching the light labels to the probe are well documented in the art. The label is measured by exciting the label and then measuring the light response with photo-detection devices. Fluorescent or phosphorescent labels can be excited by irradiation with light of the appropriate wavelength. Chemiluminescent or bioluminescent labels can be chemically excited, by methods well known in the art.

The identification probe can also be labeled with an enzyme. The hybridization product is detected by the action of the enzyme on a substrate for the enzyme. For example, an enzyme capable of acting on a chromogenic substrate can be selected. The conversion ratio of the substrate can be monitored by optical analysis. The ratio is then correlated with the presence or absence of the target polynucleotide. It is well known that the avidin and biotin can be used to link an enzyme to a specific nucleic acid probe. Examples of suitable enzymes include, for example, beta-galactosidase, alkaline phosphatase, horseradish peroxidase, and luciferase.

One of the major advantages of the light-labels and enzyme-action based labels is that their qualitative detection can be visualized without the need of expensive detection equipment. Of course, quantitative determinations of such labels can also be made with the use of photometric equipment.

It is preferable that the first and second probes each be complementary to substantially mutually exclusive portions of the target polynucleotide. In other words, the first and second probes should not compete for the same base sequence to the extent that sandwich hybridization is prevented.

The probes can be made from appropriate restriction endonuclease treated polynucleotide from the organism of interest, or from double-stranded polynucleotides by enzymatic methods such as Exo III digestion or RNA polymerase transcription. In these cases the probes are RNA or DNA fragments. In other cases where the base sequence of a unique portion is known, the probes can be synthesized by organic synthetic techniques (Stawinski, J. et al., Nuc. Acids Res. 4, 353, 1977; Gough, G. R. et al., Nuc. Acids Res. 6, 1557, 1979; Gough, G. R. et al., Nuc. Acids Res. 7, 1955, 1979; Narang, S. A., Methods in Enzymology, Vol. 65, Part I, 610-620, 1980). Also, it is possible to produce oligodeoxyribuonucleotides of defined sequence using polynucleotide phosphorylas.e (E. Coli) under proper conditions (Gillam, S., and Smith, M., Methods in Enzymology, Vol. 65, Part I, pp. 687-701, 1980). It is also possible to produce large quantities of the probe by cloning; e.g. by recombining the particular sequence into a plasmid or M13 bacteriophage vector, and then cloning the recombined moiety using standard methods. The cloning method is preferred.

The size of the probes can be from 10. nucleotides to 100,000 nucleotides in length. Below 10 nucleotides, hybridized systems are not stable and will begin to denature above 20 degrees C. A complementary polynucleotide sequence of 12 is about the minimum length required for appropriate binding specificity. The generally used minimum in practice is about 15. Above 100;000 nucleotides, hybridization (renaturation) becomes a much slower and incomplete process, see Molecular Genetics, Stent, G. S. and R. Calender, pp. 213-219, 1971. It is not necessary that the entire probe be complementary to the target polynucleotide on a base by base scale. Preferably the probes should be from about 15 to about 50,000 nucleotides long, more preferably from about 15 to about 10,000 nucleotides long. The number of complementary (base pairing) nucleotides on the probe is preferably between about 200 to about 5,000. Preferably the complementary nucleotides are adjacent to each other, especially when the number of complementary nucleotides is low (below 200). But one-to-one complementation of all of the base pairing nucleotides on the target and the probe is not necessary. Smaller probes (15-100) lend themselves to production by automated organic synthetic techniques. Probes sized from 100-10,000 nucleotides can be obtained from appropriate enzymatic methods, or by recombinant DNA methods.

The labeling of smaller polynucleotide segments with the relatively bulky labeling moieties (e.g. chemiluminescent labels) may in some cases interfere with the hybridization process. Therefore the proper choice of labels is important. Some of the criteria for choosing the labels are: ease of incorporating the label into the polynucleotide without inhibiting hybridization, ease and sensitivity of detection of the label, low nonspecific binding of the labeled probe to the solid carrier, and high stability.

The proper hybridization conditions in the solution hybridization step are determined by the nature of the first binding group on the first probe (the binding probe), and of the label attached to the second probe (the identification probe), the size of the two probes, the [G] + [C] (guanine plus cytosine) content of the probes and the complementary nucleotide sequences on the target polynucleotide, and how the test sample is prepared. The label can affect the temperature and salt concentration used for carrying out the hybridization reaction. For example, chemiluminescent catalysts can be sensitive to temperatures and salt concentrations that absorber/emitter moieties can tolerate. The size of the probes affect the temperature and time for the hybridization reaction. Assuming similar salt and reagent concentrations, hybridizations involving reagent polynucleotide sequences in the range of 10,000 to 100,000 nucleotides may require from 40 to 80 minutes to occur at 67 degrees C, while hybridizations involving 14 to 100 nucleotides require from 5 to 30 minutes at 25 degrees C. Similarly, sequences with high [G] + [C] content will hybridize at higher temperatures than polynucleotide sequences with a low [G] + [C] content. Finally, conditions used to prepare the test sample and to maintain the target polynucleotide in the single-stranded form can affect the temperature, time, and salt concentration used in the hybridization reaction. The conditions for preparing the test sample are affected by the polynucleotide length required and the [G] + [C] content. In general, the longer the sequence or the higher the [G] + [C] content, the higher the temperature and/or lower the salt concentration required for denaturation. The concentration of probe or target in the mixture also determines the time necessary for hybridization to occur. The higher the probe or target concentration the shorter the hybridization incubation time needed. The basic rate of hybridization is also affected by the type of salt present in the incubation mix, its concentration, and the temperature of incubation. Sodium chloride, sodium phosphate and sodium citrate are the salts most frequently used for hybridization and the salt concentration used can be as high as 1.5 - 2M. The salts mentioned above yield comparable rates of polynucleotide hybridization when used at the same concentrations and temperatures, as do the comparable potassium, lithium, rubidium, and cesium salts.. Britten et al. (1974) (Methods in Enzvmology, Volume XXIX, part E., ed. Grossman and Moldave; Academic Press, New York, page 364) and We tmur and Davidson (1968) (J. Molecular Biology, Vol. 31, page 349) present data which illustrates the standard basic rates of hybridization attained in commonly used salts. The hybridization rates of DNA with RNA vary somewhat from those of DNA hybridizing with DNA. The magnitude of the variation is rarely over tenfold and varies, depending for example, on whether an excess of DNA or RNA is used. See Galau et al. (1977) (Proc. Natl. Acad. Sci. USA, Vol 74, #6, pg. 2306).

There are preferred general conditions which may not be optimal, but under which hybridization occurs for nearly all probe and target combinations of 100 nucleotides or more. These conditions are about 0.75 molar sodium chloride, 0.075 molar sodium citrate, 0.025 molar sodium phosphate (pH 6.5), 65 degrees C, and polynucleotide concentrations of 0.001 to 1.0 microg./ml. There are also known methods for achieving similar conditions at lower temperatures, for example by including a denaturant such as formamide in the reaction (Casey et al., Nucl. Acids Res. 4: 1539 (1977)). Addition of other reagents in the hybridization reaction may also increase the reaction rate, for example dextran sulfate (Wahl et al., P.N.A.S. 76:3683 (1979), polyethylene glycol (Amasirro, Anal. Biochem. 152:304 (1986)) and phenol (Kohne et al., Biochemistry 16:5329 (1977)).

A kit suitable for use to detect a single stranded target polynucleotide in a test sample by the assay method of the present invention can comprise a liquid polynucleotide reagent or reagents comprising at least two different probes, being a first probe - a binding probe, and a second probe - an identification probe. Each probe comprises a single-stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide. Neither probe is affixed to a solid carrier. The probes cannot hybridize with each other. The second probe has a detectable label. The kit also comprises a solid carrier capable of binding the first probe, but not the second probe and not the target polynucleotide. The other characteristics of the probes and of the solid carrier are generally as described above in the description of the method of the present invention. The kit can also further comprise means for qualitative and/or quantitative determination of the label. There can be more than one probe in each polynucleotide reagent. For example, because the probes do not hybridize with each other, they can conveniently be combined in a single reagent.

It is also preferred that one or more of the following components be included as part of the kit: (a) detergents capable of solubilizing the various moieties, such as sodium dodecyl sulfate, or sodium lauryl sarcosinate; (b) proteases, such as proteinases K and pronase; (c) reagents to facilitate the denaturing of the target polynucleotide, including salts and solvents; (d) reagents used in detecting the label on the second probe. The components of (a), (b), (c) and the probe reagent can also be combined into a sisngle reagent or a number of reagents, as needed.

The above method and kit can be adapted for use for testing a series of target polynucleotides. A separate set of probes is provided for each target polynucleotide, each set being specific for the particular target polynucleotide. Each set of probes comprises at least two different probes, being the binding and identification probes (the first and second probes), as characterized before. The solid carrier used has a binding group capable of binding the binding groups on each of the binding probes specific for the plurality of target polynucleotides, but not any of the identification probes, and not any of the target polynucleotides. Therefore a single universal solid carrier can be used with all of the different binding probes. In the kit, preferably each set of probes is contained in a separate liquid reagent.

The above method and kit can also be adapted for simultaneous testing of several target polynucleotides in a test sample. A separate set of probes is provided for each target polynucleotide, each set being specific for the corresponding polynucleotide. All the probes are incapable of hybridizing with each other. Each set of probes consists of at least two different probes, being a first probe (a binding probe) and a second probe (an identification probe), the probes being as characterized previously. All the first probes carry an identical binding group, so that the first probes can all be harvested on the same solid carrier. The solid carrier does not bind any of the indentification probes, and not any of the target polynucleotides. The second probes comprise different labels, each label corresponding to a particular target polynucleotide. After the harvesting step, detection of the respective labels on the solid carrier yields information about the presence and/or quantities of the corresponding target polynucleotide in the sample. In the kit, preferably all of the probes are contained in a single liquid reagent.

The advantages of the assay method and kit of the present invention are many. The method gives the reaction speed and reaction efficiency of solution hybridization, and the ease of separation of the hybridization products from the test sample of solid carrier hybridization. Moreover, the assay method is highly specific, as it requires complementation of at least two probes with the target polynucleotide. The solution sandwich hybridization step can be completed generally in less than an hour, and the harvesting step takes no longer than about 5 minutes (optimally less than about 0.5 minutes). Thus substantial time savings can be obtained.

Further, the kit of the present invention is simple to use. The user need only stock one single solid carrier reagent, which can be used for assaying all target polynucleotides. Only one liquid .reagent is required for each target polynucleotide. A third reagent for detecting the label may be required in some applications.

Another significant advantage of the method of the subject invention is that no extensive preparation of the test samples is necessary. Prior art solution hybridization methods detect target nucleic acids which have been purified away from other cell components. Nucleic acids in cells and viruses are normally tightly complexed with other cell components, usually protein, and, in this form are not available for hybridization. Simply breaking the cell or virus open to release the contents does not necessarily render the polynucleotides available for hybridization. The polynucleotides remain complexed to other cell or viral components even though released from the cell, and may in fact become extensively degraded by nucleases which also may be released. In addition a probe added to such a mix may become complexed to "sticky" cell or viral components and be rendered unavailable for hybridization, or the probe may be degraded by nuclease action. A variety of prior art methods exist for purifying polynucleotides. These methods are all time consuming - one taking an hour is regarded as very rapid - and require multiple manipulations. Surprisingly, it was discovered that the assay technique of the subject invention can be used with unpurified samples, thus obviating the need for laborious and time-consuming purification steps. The assay lends itself readily to automation which could further simplify the user's task. The assay results can be either qualitative or quantitative.

Another aspect of the method and test kit of the subject invention is directed to genetic disease diagnosis and cancer diagnosis.

Ge n e t i c disease is the result of mutations in a polynucleotide's nucleotide sequence. Such mutation is also a factor in the development of cancer. Therefore, as a consequence or such genetic disease or cancer, a sample taken from the patient, and containing a normal polynucleotide (standard polynucleotide), can also contain an abnormal polynucleotide (target polynucleotide) which is a variant of the normal polynucleotide. The sample may also contain only the abnormal polynucleotide. The normal and abnormal polynucleotides typically have substantially identical nucleotide sequences, except at points of mutation on the polynucleotides. The normal and abnormal polynucleotides thus react differently to certain reagents. The method of the present invention can be used to detect such difference between the normal and abnormal polynucleotides. The method combines a severing step with a solution sandwich hybridization procedure. The severing step comprises a segmenting step, and may also comprise a denaturing step if the target polynucleotide is double stranded. The order of these two steps is not significant.

Restriction reagents, e.g., restriction enzymes, are known. A restriction enzyme can, under the proper conditions, be used in a segmenting step to divide a polynucleotide at very specific sites on the nucleotide backbone. The standard and target polynucleotides are substantially similar. The standard polynucleotide has a portion A and a portion B. The target polynucleotide has a portion A' and a portion B'. The two polynucleotides differ by at least one nucleotide which is located between the polynucleotides' respective portions A and B, and A' and B'. The restriction reagent is chosen such that it segments the standard polynucleotide into segments none of which contains both portions A and B; the restriction reagent segments the target polynucleotide into segments at least one of which contains both portions A' and B'. In the typical case, where the target polynucleotide is a genetic variant of the standard polynucleotide, portions A and A', and portions B and B', are identical.

The target and standard polynucleotides present, or their segments (from the segment step), are denatured, i.e. rendered single-stranded, if necessary, either before or after the segmenting step. The treated sample thus contains single-stranded versions of the target and/or standard polynucleotides present, ready for hybridization. The treated sample is then combined with at least two probes to form a reaction mixture. The two probes are a first probe and a second probe. Each probe comprises a singlestranded polynucleotide. The probes do not hybridize with each other. The first probe complementary base pairs with portion A on the. standard polynucleotide, and with portion A' on the target polynucleotide. The second probe complementary base pairs with portion B on the standard polynucleotide, and with portion B' on the target polynucleotide. The two probes together form hybrid molecules by base pairing with the single-stranded segment(s) of the target polynucleotide which contains both portions A' and B'. With respect to the standard polynucleotide, because portion A and portion B are on separate segments after sequentation, no hybrid molecules incorporating both the first and second probes will form. The probes are attached to separate segments. Hybrid molecules having both the first and second probes will form only if the abnormal (target) polynucleotide is present in the sample.

The presence in the reaction mixture of hybrid molecules incorporating both of the probes would indicate the presence of the target polynucleotide in the sample. The determination of the hybrid molecules formed by this sandwich hybridization procedure can be performed using previously known methods. For example, a system similar to that disclosed in Ranki, U.S. Patent No. 4,486,539, wherein one of the probes is already immobilized on a solid carrier, can be used. That is, not all of the probes need to be in solution. Other methods are also known. Alternatively, solution sandwich hybridization, followed by a harvesting step, as previously discussed can also be used. In the latter case, the further limitations on the method of this invention as described above are that: neither probe is affixed to a solid carrier, and the second probe has a detectable label. The reaction mixture is contacted with a solid carrier which binds the first probe, but not the second probe, and not any of the segments containing only portion B or only portion B'. A subsequent determination is made to see if any of the label is bound on the solidcarrier, as previously discussed.

For example, the following method is suitable for sickle cell assays. A test sample suspected to contain the mutated gene DNA strands (e.g. in sickle alleles) is treated with a restriction enzyme, e.g. Ms t II. Such enzymes have been used in gene mapping, in order to detect structural gene deletions in the DNA strands. Direct identification of mutant genes in DNA, e.g. hemoglobinopathies due to point mutation in the DNA, was also possible by virtue of the specificity of such restriction enzymes. A single nucleotide change in an enzyme's cleavage site can readily be detected if the appropriate enzyme is used. It is known that the restriction enzyme Mst II cleaves DNA on the average to produce fragments of 1.1 and 1.3 kb from β^N and β⁵ genes, respectively. Mst II cleaves a sequence (CCTNAGG) that is a subset of Ddel sites (CTNAG). The DNA from the sickle cell gene has a nucleotide variation at the Mst II site. Therefore, the mutated DNA strand will not be cleaved at that site by the Mst II enzyme. The restriction enzyme treated test sample is then subject to conditions which renders the polynucleotides single stranded. A single target polynucleotide-single sample hybridization assay procedure as previously described for detecting the presence of a target polynucleotide would then be carried out, with the additional limitation that the first and second probes bind sites on the DNA strand which are oh opposite sides of the Mst II site involved in the mutation. The only way that a complex hybrid molecule, comprising the target polynucleotide and both of the probes, will form, is if the gene is a mutated one - e.g. a sickle cell. The normal gene is cleaved between the binding sites for the two probes. The schematic of this sickle cell assay is represented in Fig. 2. This method eliminates the prior art assay steps of gel electrophoresis and transfer to solid support (filter paper). The simplification is very substantial. This assay procedure would apply equally well to the diagnosis of other genetic diseases in which gene mutations are involved.

The cancer diagnostic aspect would take the form of a quantitative test for the levels of messenger RNA from an oncogene, although tests similar to the sickle cell example would also be useful.

EXAMPLES

EXAMPLE 1

This example correlates the harvested signal to the target DNA concentration. In this example the target polynucleotide is mpB1017; probe 1 is pHBC6 having biotin as the first binding group; probe 2 is M13 10W labeled with ³²p; the solid carrier is avidin-cellulose, which binds biotin on the first probe.

ABBREVIATIONS. SSC is 0.15 molar sodium chloride and 0.015 molar sodium citrate. SSC is used at various concentrations, "10X SSC" would be, for example, 1.5 molar sodium chloride, and 0.15 molar sodium citrate. EDTA is ethylenediamine tetra-acetate, SDS is sodium dodecyl sulfate, DNA is deoxribopolynucleotide, BSA is bovine serum albumin, tris:HCL is tris-hydroxymethylaminomethane adjusted to the appropriate pH with hydrochloric acid.

MATERIALS AND METHODS. Plasmid DNAs were obtained by growing transformed E. coli, strain LE392, followed by lysis of the cells with lysozyme, SDS and NaOH. DNA was further purified by centrif ugation in CsCl in the presense of ethidium bromide See Mauiatis, T., Fritsch, E. F., and Sambrook, J., 1982. Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, U.S.A. Replicative form (RF) DNA from M13 bacteriophage, strain 10w was obtained in an identical fashion by growing infected E. coli strain 71.18. Single-stranded virion DNA from M13 clone mpB1017 was obtained from virions in the growth media of infected E. coli 71.18 by precipitation with polyethylene glycol, centrifugation in CsCl, and extraction of the purified virus with SDS, phenol, and chloroform. Salmon, testes DNA was obtained from Sigma Chemical Company, St. Louis, MO (USA).

Double-stranded DNAs were labeled by nick- translation (Rigby, et al., J. Mol. Biol., 113:237, 1977) essentially as described by Leary et al., Proc. Nat'l. Acad. Sci. [USA] 80:4045, 1983. Plasmid pHBC6 contains 5,400 base pairs of the human beta-globin gene, cloned in the plasmid pBR322 (Fukumaki et al., Cell 28:58.5, 1982). M13 clone mpB1017 contains 1310 bases of the human globin gene, homologous to 1310 bases from the plasmid pHBC6. M13 strain 10w contains 7,500 bases of bacteriophage genes, homologous to the same number of bases in clone mpB1017. The regions in mpB1017 DNA that are homologous to pHBC6 and M13 10w are not overlapping.

The solid phase for harvesting the solution- sandwich product was prepared by activation of microcrystalline cellulose with N,N'-carbonyldiimidazole, followed by coupling to the protein avidin, using methods similar to those of Paul et al., J. Org. Chem. 27:2094, 1962. The coupling ratio was 1 gram of activated cellulose to 1 mg of avidin. A 1:1 slurry of the prepared solid phase of avidin:cellulose contains about 1 gm of cellulose in 6 ml of buffer.

Solution hybridizations were preformed in a final solution containing 5X SSC, 0.02% (w/v) each of polyvinylpyrolidone-40, ficoll-400, and BSA, as well as 25 mM NaPO₄ buffer, pH 6.5, and 250 microg./ml sonicated salmon testes DNA. All DNAs (probe 1, probe 2, target, and carrier salmon testes) were denatured at 100 degrees C for 3 to 5 minutes prior to starting the hybridization reaction.

Further details of the example assay are given below.

PROCEDURE. The following assay was done to examine the feasibility of the assay of; the present invention, and to determine the range of target DNA concentration in which the assay would be effective. Plasmid pHBC6 was labeled with the ligand biotin by nick-translation with biotin-11- deoxyuridine triphosphate (Bethesda Research Laboratories, Gaithersburg, MD, USA). This constitutes probe 1 of the assay. The replicative form DNA of bacteriophage M13 10w was labeled with a radioactive reporter, ³² P, by nick- translation with alpha-³²P-deoxycytidine triphosphate (Amersham, Chicago, IL, USA). This constitutes probe 2 of the assay. Probe one contained approximately 6% biotin nucleotides, and probe 2 had specific radioactivities of 0.7 to 7 X 10⁷ cpm/microg. The target DNA was M13 mpB1017, and samples containing a range of amounts of target DNA from 20 picogra ms to 1.0 microgram were teste d. All as says conta ined 100 ng of probe 1 ( b iot in) and 50 ng o f probe 2

(³² P). Probe and sample DNAs were mixed together in a solution of 10 mM tris:HCl, pH7.5, 1.0 mM EDTA, heat denatured, and adjusted to the solution hybridization conditions given in the methods above and incubated in a total volume of 0.2 ml overnight at 68 degrees C. This incubation time is excessive, and was used only for convenience and to assure completeness of the reaction. Control assays contained probe 1 or probe 2 and 20 ng. of target, or probe 1 & probe 2 without target. The separation of the product sandwich from unhybridized probes was accomplished by adding 0.2 ml of a 1:1 slurry of avidin:cellulose to the reactions, incubating at 22 degrees C for 30 min., and filtering the cellulose onto a 13 mm glass fiber filter. One wash of the reaction vessel with 0.1 ml. of tris:HCl, pH 7.5, 1 mM ED-TA was also filtered on the same filter as the primary reaction. The filtered solid support was further washed 2 times with 2X SSC and 0.1% (v/v) SDS, 2 times with 0.2X SSC and 0.1% SDS, and 2 times with 0.1X SSC and 0.1% SDS. The last two washes were performed at 50 degrees C, all others at room temperature. All washes were with 0.5 ml of solution. These washes were adapted from those used for Southern blot hybridization analysis by Leary et al., P.N.A.S. 80:4045 (1983)) The amount of probe 2 (^{3 2}P) bound to the solid support was determined by liquid scintillation counting, with the resin in 0.5 ml of solution in a minivial and 4.5 ml of Beckman Ready Solv _TM MP scintillation fluid. All washes were also collected and counted similarly. RESULTS. The results from one run are given in Table I, where the amount of probe 2 bound is related directly to the amount of target DNA in the assay. The data represent the average of duplicate samples.

Data from several runs demonstrated that the assay as performed in this example has a peak of harvested signal (probe 2) at about 30 ng of target DNA, at amounts above this level, the harvested signal decreases in a logarithmic fashion. Below 30 ng of target DNA, the assay is limited by background binding of probe 2 to the solid support and the specific radioactivity of the probe, but increases exponentially from about 0.5 ng to 10 ng of target. The non-linear nature of the signal as a function of the target amount is graphically depicted in Figure 3. Fig. 4 is an expanded version of Fig. 3 around the peaked signal. The non-linear relationship may be a function of the nicktranslation reaction used to label the probes, since probes labeled in this fashion are known to show some elements of cooperative binding (Meinkoth and Wahl, Anal. Biochem. 138:267, 1984).

EXAMPLE 2

This example demonstrates the binding characteristics of an avidin-cellulose solid carrier and a biotin-labeled probe.

The kinetics and specificity of harvesting a biotin-labeled probe by binding it out of a hybridization buffer onto avidin-cellulose were examined using DNA's labeled with both biotin-11-dUTP and ³H-dATP or labeled with ³H-dATP alone (control). The DNA was plasmid pHBC-6 labeled by nick-translation (Rigby et al., J. Md. Biol., 113:237 (1977)).

Avidin-cellulose (0.05 ml packed volume) suspended in 0.2 ml of 10mM Tris:HCl plus 1.0 mM EDTA (pH 7.5) was placed in each of 30 microcentrifuge tubes. A mixture (0.2 ml) containing 100 nanogm. of the biotin-labeled or control DNA, 50 microgm. of carrier salmon testes DNA, 20 microgm. of yeast RNA, 0.02% (w/v) each of bovine serum albumin, ficoll, and polyvinylpryolidone in 0.075 M sodium citrate, 0.75 M sodium chloride, and 0.025 M sodium phosphate (pH 6.5) was added to the appropriate tubes. The tubes containing the solid phase and hybridization mixture were incubated at room temperature on a rocking platform for times ranging from 1 min. to 300 min. After the incubation, the solid phase was collected by filtration on a teflon membrane filter. The fraction of DNA bound was determined by subtracting the radioactivity in the filtrate and washes from the total radioactivity added to each sample. The dat a are presented in Figure 5. Within 30 minutes, approximately 92% of the biotin-labeled DNA became bound to the avidincellulose, while an average of less than 2% of the control DNA bound to the solid carrier. The binding is rapid, efficient, and specific for the biotin label on the probe.

The requirement for the avidin component of the solid phase for binding to biotin-labeled DNA was examined using the same DNA's as above, with various pretreatments of the solid carrier. Free biotin should compete with biotin-DNA for the avidin sites on the solid phase, and pretreatment of the solid phase with 20 times the half-capacity of free biotin reduced biotin-DNA binding by 50%. A further pretreatment with 10 fold more free biotin reduced biotin-DNA binding by an additional 30%. Pretreatment of the solid phase with pronase, 1% (w/v) sodium dodecyl sulfate and 0.1% (v/v) beta- raercaptoethanol also reduced biotin-DNA binding by 50%, and further treatment of this digested resin with free biotin abolished biotin-DNA binding. A slightly different approach also indicated that the large majority of biotin-DNA binding to the solid carrier was not only biotin-dependent but also avidin-dependent. A control solid carrier was prepared by treating cellulose in the same manner as used for coupling to avidin, except that the avidin was left out of the reaction and replaced with Tris:HCl. This control resin bound 5 to 6% of control DNA probe and 15 to 16% of biotinlabeled DNA probe under the conditions used above for the kinetics study. Thus, the majority of biotin-labeled probe binding (larger than 85%) to the solid carrier is due to interaction with the specific ligand avidin.

EXAMPLE 3

This example demonstrates the construction of M13 vectors containing Mst II fragments bordering the sicklecell mutation.

In order to assay the major genetic form of sickle-cell anemia using the assay method of the present invention, very specific M13 constructions are required. Basically, DNA fragments from either side of the Mst II restriction site which identifies the 6th codon error in a sickle-cell individual were inserted into the M13 phage. The two fragments used for this purpose were derived from the plasmid pHBC6 (Fukumaki et al., Cell 28: 585 (1982)) and purified by acrylamide gel electropKoresis. Approximately 10 ug of the 200 base pair Sau 1 (Mst II isoschizomer) located immediately downstream of the diagnostic Mst II site was treated with Klenow DNA Polymerase in the presence of 250 uM dXTPs to "fill-in" the Mst II site. This "blunt" DNA fragment was then inserted into the He II site of pUC 18. Analysis of 24 pUC 18-B-200 isolates indicated the presence of the 200 base insert in 2 isolates, pUC 18-B-200-6 and pUC 18-B-200-9. Following CsCl preparation, approximately 200 ug of pϋC 18-B-200-9 was digested with Eco R1 and Hd III prior to purification by gel electrophoresis. This Eco R1, Hd III ended 200 base pair fragment was then inserted into M13 mp 18 to obtain single-stranded DNA. Additional constructions were made using this 200 base fragment to allow simple production of large quantities of specific RNA sequences. Specifically, this fragment was inserted into the transcription vector pT7-1 and pT7-2 (United States Biochemical Corporation, P.O. Box 22406, Cleveland, Ohio, 44122). These constructions allow for the in vitro synthesis of labelled RNA to use as a probe in the sickle- cell assay of the present invention. The other recombinant vectors needed for assay of the sickle-cell trait use the 800 base pair Mst II - Hpa 1 fragment located immediately upstream of the diagnostic Mst II restriction site. This fragment was cloned in a similar fashion again using Klenow DNA Polymerase to "round-off" the 5' overhanging ends found at the Mst II end. This 800 base pair fragment was inserted into four vectors in order to obtain single stranded RNA and DNA. They are:

M13 B-19-1-800 β -globin message-like strand M13 B-18-1-800 β -globin non-message-like

PT7-1-800 RNA which is non-message like pT7-2-800 RNA which is message-like

Although the present invention has been described in considerable detail with regard to certain versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not necessarily be limited to the description of the versions contained herein.

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC

(51) International Patent Classification 4 : (11) International Publication Number : WO 88/ 0 C12Q 1/68 A3 (43) International Publication Date: 21 April 1988 (21.

(21) International Application Number: PCT/US87/02548 (74) Agents: MAY, William, H. et al. ; Beckman I ments, Inc., 2500 Harbor Boulevard, Fullerton

(22) International Filing Date: 6 October 1987 (06.10.87) 92634 (US).

(31) Priority Application Number: 919,201 (81) Designated States: AT (European patent), AU, BE ropean patent), CH (European patent), DE (

(32) Priority Date: 14 October 1986 (14.10.86) pean patent), DK, FI, FR (European patent), (European patent), IT (European patent), JP,

(33) Priority Country: US (European patent), NL (European patent), N (European patent).

(71) Applicant: BECKMAN INSTRUMENTS, INC. [US/ Published

US]; 2500 Harbor Boulevard, Fullerton, CA 92634 With international search report. (US). Before the expiration of the time limit for amendin claims and to be republished in the event of the recei

(72) Inventors: BEEBE, Joan, Marlyn ; 3811 Glen Ridge

Drive, Chino Hills, CA 91709 (US). GLANVILLE, amendments. Linda, Lee ; 10340 Pharlap Drive, Cupertino, CA 95014 (US). LEARY, Jeffry, Joseph ; 520 Cherry (88) Date of publication of the international search report: Street, Brea, CA 92621 (US). RICE, Edward, Gray ; 14 July 1988 (14.0 827 Ross Court, Palo Alto, CA 94303 (US).

(54) Title: IMPROVED NUCLEIC ACID HYBRIDIZATION TECHNIQUE AND KIT THEREFOR

(57) Abstract

A method for detecting a polynucleotide in a sample comprising: combining in liquid phase the sample with a and second probe, each binding to different sequences of the target polynucleotide. The "target/probe" complex is im ilized subsequently with a solid carrier able to bind the first probe. The second probe carries a detectable label. A kit use with the method and the use thereof for the diagnosis of genetic diseases, such as sickle cell anaemia, and for canc disclosed.

Claims

What is claimed is:

1. An assay method for detecting a single- stranded target polynucleotide in a liquid sample, the method comprising the steps of:

(b) subsequently contacting the reaction mixture with a solid carrier which binds the first probe but not the second probe and not the target polynucleotide; and

(c) subsequently determining if any of the label is bound on the solid carrier.

2. The method of claim 1 wherein the first probe is a RNA or DNA fragment.

3. The method of claim 1 wherein the second probe is a RNA or DNA fragment.

4. The method of claim 1 wherein the solid carrier is formed of a material selected from the group consisting of agarose, cellulose, glass, latex. polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof.

5. The method of claim 1 wherein the solid carrier is in a form selected from the group consisting of micro-particulates, macro-particulates, sheets, beads, tubes, pipet tips, plates, and filters.

6. The method of claim 1 wherein the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes,enzyme cofactors, hapten , antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof.

7. The method of claim 1 wherein the first probe has a first binding group and the solid carrier has a second binding group, the two binding groups capable of binding each other, the two binding groups constituting a binding pair.

8. The method of claim 7 wherein the binding pair is selected from the group consisting of avidin-biotin, haptens-antibodies, antibodies-antigens, carbohydrateslectins, riboflavin-riboflavin binding protein, metal ionsmetal ion binding substances, enzymes-substrates, boronatescis dioles, staph A proteins-antibodies, enzymes-inhibitors, and derivatives thereof.

9. The method of claim 8 wherein the binding pair is biotin-avidin, with the solid carrier being avidincellulose.

10. The method of claim 1 in which each probe is between about 15 to about 50,000 nucleotides long, the first and second probes each being complementary to different portions of the target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

11. The method of claim 10 wherein the portions complementary to the probes are immediately adjacent.

12. The method of claim 10 in which the first probe is a RNA or DNA fragment, and the second probe is a RNA or DNA fragment; the solid carrier is formed of a material selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof; the solid carrier is in a form selected from the group consisting of micro-particulates, macro-particulates, sheets, beads, tubes, pipet tips, plates, and filter; the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof; the first probe has a first binding group and the solid carrier has a second binding group, the two binding groups capable of binding each other; the two binding groups constituting a binding pair, the binding pair being selected from the group consisting of avidin-biotin, haptens-antibodies, antibodies- antigens, carbohydrates-lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzymes-substrates, boronates-cis dioles, staph A proteins- antibodies, enzymes-inhibitors, and derivatives thereof.

13. The method of- claim 1 wherein the detection of the target polynucleotide is qualitative.

14. The method of claim 1 wherein the presence of the label. bound on the solid carrier is determined quantitatively, such that the detection of the target polynucleotide is quantitative.

15. The method of claim 1 wherein the probes are combined with the sample in seriatum.

16. The method of claim 1 wherein the sample is combined with all the probes simultaneously.

17. The method of claim 1 wherein the single stranded target polynucleotide is formed by denaturing a double-stranded polynucleotide.

18. The method of cl a i m 1 wherein the step for determining if any of the label is bound on the solid carrier further comprises the steps of:

(a) separating the second probe which is not incorporated in the hybrid molecules from the solid carrier; and

(b) subsequently detecting the presence or absence of the label on the solid carrier, the presence of w hich indicates the presence of the target polynucleotide in the sample.

19. The method of claim 1 wherein the step for determining if any of the label is bound on the solid carrier further comprises the steps of: (a) measuring the amount of unbound second probe in solution; and

(b) comparing that to the total amount of second probe added to the reaction mixture to determine the presence or absence of the target polynucleotide in the sample.

20. An assay method for detecting a plurality of single-stranded target polynucleotides in a plurality of liquid samples, for each target polynucleotide, the method comprising the steps of:

(a) combining at least a portion of a liquid sample suspected to contain the target nucleic acid with at least two different probes, being a first probe and a second probe, to form a reaction mixture, each probe comprising a single-stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the first probe and second probe together forming hybrid molecules by complementary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the probes do not hybridize with each other. and the second probe having a detectable label;

(b) subsequently contacting the reaction mixture with a solid carrier which binds the first probe but not the second probe and not any of the target polynucleotides; and

(c) subsequently determining if any of the label is bound on the solid carrier;

wherein the solid carrier binds each of the first probes specific for the plurality of target polynucleotides.

21. The method of claim 20 wherein each of the first probes has a first binding group and the solid carrier has a second binding group, the second binding group capable of binding each of the first binding groups on the first probes speciific for the plurality of target polynucleotides.

22. The method of claim 20, in which each probe is between about 15 to about 50,000 nucleotides long; for each pair of probes which is specific for each target polynucleotide, the first and second probes being complementary to different portions of the respective target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

23. The method of claim 22 wherein the portions are immediately adjacent.

24. The method of claim 20 wherein at least one of the single-stranded target polynucleotides is formed by denaturing a double stranded polynucleotide.

25. The method of claim 20 wherein at least some of the probes are combined with the sample at different times.

26. An assay method for detecting a plurality of single stranded target polynucleotides in a liquid sample, the method comprising the steps of:

(a) combining the sample with a plurality of different probes to form a reaction mixture, at least two of the probes being specific for each of the target polynucleotides, being a first probe and a second probe, the first probe and second probe each comprising a single stranded polynucleotide which contains a nucleotide seqence complementary to a portion of the respective target polynucleotide, the first probe and second probe together forming hybrid molecules by complementary base pairing with the target poluynucleotide, the second probe having a detectable label; wherein none of the plurality of probes is affixed to a solid carrier, the plurality of probes do not hybridize with each other, and each of the second probes specific for the plurality of target polynucleotides has a different label;

(b) subsequently contacting the reaction mixture with a solid carrier which binds the first probes but not the second probes and not any of the target polynucleotides; and

(c) subsequently determining if any of each of the different labels is bound on the solid carrier.

27. The method of claim 26 in which each probe is between about 15 to about 50,000 nucleotides long, the first probe and second probe specific for each target polynucleotide each being complementary to different portions of the respective target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides apart.

28. The method of claim 27 wherein the portions are immediately adjacent.

29. The method of claim 26 wherein at least one of the sing l e-stranded target polynucleotides is formed by denaturing a double-stranded polynucleotide.

30. The method of claim 26 wherein at least some of the plurality of probes are combined with the sample at different times.

31. The method of claim 26 wherein all of the probes are siumltaneously combined with the sample.

32. A kit for the detection of a single-stranded target polynucleotide in a sample, the kit comprising:

(a) polynucleotide reagents compris ing a t least two different probes , being a f irst probe and a second probe, each probe comprising a single-stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the probes together forming hybrid molecules by co mplemen tary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the probes can not hybridize with each other, the second probe having a detectable label; and

(b) a solid carrier which binds the first probe, but not the second probe and not the target polynucleotide.

33. The kit of claim 32 wherein the first probe is a RNA or DNA fragment.

34. The kit of claim 32 wherein the second probe is a RNA or DNA fragment.

35. The kit of claim 32 wherein the solid carrier is formed of a material selected from the group consisting of agarose, cellulose, glass, latex, nylon, polyacrylamide, polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silca, and derivatives thereof.

36. The kit of claim 32 wherein the solid carrier is in a form selected from the group consisting of microparticulates, macro-particulates, sheets, beads, tubes, pipet tips, plates, and filters.

37. The kit of claim 32 wherein the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof.

38. The kit of claim 32 wherein the first probe has a first binding group and the solid carrier has a second binding group, the two binding groups capable of binding to each other, the two binding groups constituting a binding pair.

39. The kit of claim 38 wherein the binding pair is selected from the group consisting of avidin-biotin, haptens-antibodies, antibodies-antigens, carbohydrateslectins, ribo flavin-ribo flavin binding protein, metal ionsmetal ion binding substances, enzymes-substrates, boronates- cis dioles, staph A proteins-antibodies, enzymes-inhibitors, and derivatives thereof.

40. The kit of claim 39 wherein the binding pair is biotin-avidin, with the solid carrier being avidin- cellulose.

41. The kit of claim 32 in which each probe is between about 15 to about 50,000 nucleotides long, the first and second probes each being complementary to different portions of the target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

42. The kit of claim 41 wherein the portions complementary to the probes are immediate ly ad jacen t .

43. The kit of claim 32 further comprising means for qualitatively detecting the label.

44. The kit of claim 32 further comprising means for quantitatively detecting the label.

45. The kit of claim 32 wherein at least one of the polynucleotide reagents contains more than one probe.

46. The kit of claim 32 wherein all of the probes are in a single reagent.

47. A kit for the detection of a plurality of single-stranded target polynucleotides, the kit comprising, for each target polynucleotide, polynucleotide reagents comprising at least two different probes, being a first probe and a second probe, each probe comprising a singlestranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the first probe and second probe together forming hybrid molecules by complementary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the probes can not hybridize with each other, the second probe having a detectable label;

the kit further comprising a solid carrier capable of binding each of the first probes, but not any of the second probes, and not any of the target polynucleotides,

48. The kit of claim 47 wherein each of the first probes has a first binding group and the solid carrier has a second binding group, the second binding group capable of binding each of the first binding groups on the first probes.

49. The kit of claim 47 wherein each second probe specific for each target polynucleotide comprises a different label.

50. The kit of claim 49 further comprising means for detecting the labels on the second probes.

51. The kit of claim 48 in which each probe comprises a single-stranded polynucleotide about 15 to about 50,000 nucleotides long, for each pair of probes specific to a target polynucleotide, each of the first and second probes being complementary to a different portion of the target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

52. The kit of claim 51 wherein the probes are complementary to immediately adjacent portions on the target polynucleotide.

53. The kit of claim 47 wherein at least one of the polynucleotide reagents contains more than one probe.

54. The kit of claim 47 wherein all of the probes complementary to each of the target polynucleotides are contained in a single reagent, the number of polynucleotide reagents in the kit therefore being equal to the number of target polynucleotides.

55. A kit for the detection of a plurality of target polynucleotides in a sample, the kit comprising:

(a) polynucleotide reagents comprising, for each of the target polynucleotides, at least two different probes, being a first probe and a second probe, each probe comprising a single-stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the first probe and second probe together forming hybrid molecules by complementary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the first probe and second probe can not hybridize with each other, the second probe having a detectable label; and wherein all of the probes for the plurality of target polynucleotides are incapable of hybridizing with each other, and wherein each of the second probes specific for the plurality of target poynucleotides has a different detectable label; and

(b) a solid carrier which binds the first probes, but not any of the second probes, and not any of the target polynucleotides.

56. The kit of claim 55 further comprising means for detecting the different labels on the second probes.

57. The kit of claim 56 in which each probe comprises a single-stranded polynucleotide about 15 to about 50,000 nucleotides long, for each pair of probes specific to each target polynucleotide, the first and second probes each being complementary to a different portion of the targe t polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

58. The method of claim 57 wherein the first and second probes are complementary to immediately adjacent portions on the target polynucleotide.

59. The kit of claim 55 wherein at least one of the polynucleotide reagents contains more than one probe.

60. The kit of claim 55 wherein all of the probes are contained in a single reagent.

61. An assay method for detecting a target polynucleotide in a sample which may also contain a standard polynucleotide, the nucleotide sequences of the two polynucleotides being substantially sirnilar, the standard polynucleotide having a portion A and a portion B, the target polynucleotide having a portion A' and a portion B', the standard and target polynucleotides differing in at least one nucleotide which is located between portions A and B on the standard polynucleotide and between portions A' and B' on the target polynucleotide, the method comprising the steps of:

62. The method of claim 61 wherein the severing, step comprises treatment with a restriction enzyme.

63. The method of claim62 wherein the target and standard polynucleotides are double-stranded, and the severing step also comprises denaturation of the. target and standard polynucleotides present.

64. The method of claim 61 wherein portion A is identical to portion A', and portion B is identical to portion B'.

65. The method of claim 61 wherein the shortest distance between portion A and portion B on the standard polynucleotide, and the shortest distance between portions A' and B' on the target polynucleotide, are no more than about 300 nucleotides.

66. The method of claim 65 wherein the shortest distance between portion A and portion B on the standard polynucleotide, and the shortest distance between portions A' and B' on the target polynucleotide, are no more than about 10 nucleotides.

67. The method of claim 61 wherein the target polynucleotide is a genetic variant of the standard polynucleotide, the target polynucleotide being the result of gene mutation which. is indicative of a genetic disease.

68. The method of claim 67 wherein the genetic disease is sickle cell anemia.

69. The method of claim 61 wherein neither probe is affixed to a solid carrier, the second probe having a detectable label, the step for determining the presence of hybrid molecules containing both of the probes further comprising the steps of:

(a) contacting the reaction mixture with a solid carrier which binds the first probe, but not the second probe, and not segments containing only portion B or portion B'; and

(b) subsequently determining if any of the label is bound on the solid carrier.

70. The method of claim 69 wherein the solid carrier is formed of a material selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide, polycarbonate, polyethylene. polypropylene, polystyrene, silica gel, silica, and derivatives thereof.

71. The method of claim 69 wherein the solid carrier is in a form selected from the group consisting of micro-particulates, macro-particulates, sheets, beads, tubes, pipet tips, plates, and filters.

72. The method of claim 69 wherein the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof.

73. The method of claim 69 wherein the first probe has a first binding group and the solid carrier has a second binding group, the two binding groups capable of binding each other, the two binding groups constituting a binding pair.

74. The method of claim 73 wherein the binding pair is selected from the group consisting of avidin-biotin, haptens-antibodies, antibodies-antigens, carbohydrateslectins, riboflavin-riboflavin binding protein, metal ionsmetal ion binding substances, enzymes-substrates, boronatescis dioles, staph A proteins-antibodies, enzymes-inhibitors, and derivatives thereof.

75. The method of claim 74 wherein the binding pair is biotin-avidin, with the solid carrier being avidin- cellulose.

76. The method of claim 69 in which each probe is between about 15 to about 50,000 nucleotides long, the first and second probes each being complementary to different portions of the target polynucleotide, the shortest distance between the portions being no more than about 300 nucleotides.

77. The method of claim 76 wherein the portions complementary to the probes are immediately adjacent.

78. The method of claim 76 in which the first probe is a RNA or DNA fragment, and the second probe is a RNA or DNA fragment; the solid carrier is formed of a material selected from the group consisting of agarose, cellulose, glass, latex, polyacrylamide, polyamide,. polycarbonate, polyethylene, polypropylene, polystyrene, silica gel, silica, and derivatives thereof; the solid carrier is in a form selected from the group consisting of micro-particulates, macro-particulates, sheets, beads, tubes, pipet tips, plates, and filter; the label is selected from the group consisting of radioisotopes, chemiluminescent compounds, bioluminescent compounds, fluorescent compounds, phosphorescent compounds, enzymes, enzyme cofactors, haptens, antibodies, avidin, biotin, carbohydrates, lectins, metal chelators, and derivatives thereof; the first probe has a first binding group and the solid carrier has a second binding group, the two binding groups capable of binding each other; the two binding groups constituting a binding pair, the binding pair being selected from the group consisting of avidin-biotin, haptens-antibodies, antibodiesantigens, carbohydrates-lectins, riboflavin-riboflavin binding protein, metal ions-metal ion binding substances, enzymes-substrates, boronates-cis dioles, staph A proteinsantibodies, enzymes-inhibitors, and derivatives thereof.

79. The method of claim 69 wherein the step for determining if any of the label is bound on the solid carrier further comprises the steps of:

(b) subsequently detecting the presence or absence of the label on the solid carrier, the presence of which indicates the presence of the target polynucleotide in the sample.

80. The method of claim 69 wherein the step for determining if any of the label is bound on the solid carrier further comprises the steps of:

(a) measuring the amount of unbound second probe in solution; and